Two Carotenoid Oxygenases Contribute to Mammalian Provitamin A Metabolism

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15′-oxygenase (BCO1) and the β-carotene-9′,10′-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15′-apocarotenoids, C₂₀), whereas eccentric cleavage by BCO2 produces long-chain (>C₂₀) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1^−/−, Bco2^−/−, and Bco1^−/−Bco2^−/− double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10′-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

Conversion of carotenoids into vitamins A(1) and A(2) in two species of freshwater fish

1. Examination of two zooplankton species predominating in fish ponds, Daphnia magna and Chironomus larvae, revealed the presence of α- and β-carotene, echinenone, canthaxanthin and 3-hydroxy-4-oxo-β-carotene in Daphnia, and β-carotene and cryptoxanthin ester in Chironomus. No specific provitamins A₂ (containing a 3,4-dehydro-β-ionone ring) were detected. 2. Guppies (Lebistes reticulatus) and platies (Xiphophorus variatus) were found to form vitamin A from β-carotene and from its oxygen-containing derivatives isozeaxanthin, canthaxanthin and astaxanthin. Slight conversion into vitamin A₂ seemed to occur simultaneously. 3,4-Dehydro-3′-hydroxy-β-carotene formed little vitamin A, and the latter was mainly of the A₂ type. Lutein was devoid of provitamin A properties. 3. In addition to vitamin A, β-carotene was detected in fish receiving the 4-oxo- and 4-hydroxy-carotenoids. A reaction scheme for the conversion of carotenoids into retinal and and 3,4-dehydroretinal is presented. 4. It is concluded that natural 4-oxo derivatives of β-carotene may play a significant role as vitamin A precursors for fish.     

Longitudinal Survey of Carotenoids in Human Milk from Urban Cohorts in China,Mexico, and the USA

Emerging evidence indicates that carotenoids may have particular roles in infant nutrition and development, yet data on the profile and bioavailability of carotenoids from human milk remain sparse. Milk was longitudinally collected at 2, 4, 13, and 26 weeks postpartum from twenty mothers each in China, Mexico, and the USA in the Global Exploration of Human Milk Study (n = 60 donors, n = 240 samples). Maternal and neonatal plasma was analyzed for carotenoids from the USA cohort at 4 weeks postpartum. Carotenoids were analyzed by HPLC and total lipids by Creamatocrit. Across all countries and lactation stages, the top four carotenoids were lutein (median 114.4 nmol/L), β-carotene (49.4 nmol/L), β-cryptoxanthin (33.8 nmol/L), and lycopene (33.7 nmol/L). Non-provitamin A carotenoids (nmol/L) and total lipids (g/L) decreased (p<0.05) with increasing lactation stage, except the provitamin A carotenoids α- and β-cryptoxanthin and β-carotene did not significantly change (p>0.05) with lactation stage. Total carotenoid content and lutein content were greatest from China, yet lycopene was lowest from China (p<0.0001). Lutein, β-cryptoxanthin, and β-carotene, and lycopene concentrations in milk were significantly correlated to maternal plasma and neonatal plasma concentrations (p<0.05), with the exception that lycopene was not significantly associated between human milk and neonatal plasma (p>0.3). This enhanced understanding of neonatal exposure to carotenoids during development may help guide dietary recommendations and design of human milk mimetics.     

An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability,bioconversion, and vitamin A status in humans

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [¹³C₁₀]β-carotene and 1 mg [¹³C₁₀]retinyl acetate in human subjects over a 2 week period. A reverse phase C₁₈ column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [¹²C]β-carotene and [¹²C] retinoids; m/z 547→330 and m/z 274→98 for [¹³C₁₀]β-carotene and [¹³C₅] cleavage products; and m/z 279→100 for metabolites of [¹³C₁₀]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [¹³C₁₀]retinyl acetate with [¹³C₁₀]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual''s vitamin A status.     

Characterization of the Role of β-Carotene 9,10-Dioxygenase in Macular Pigment Metabolism

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation.     

The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').     

Biological actions of carotenoids

Of 600 carotenoids from natural sources that have been characterized, fewer than 10% serve as precursors of vitamin A. Many dietary carotenoids, both with and without provitamin A activity, are found in the blood and tissues of humans. beta-Carotene, the most nutritionally active carotenoid, comprises 15-30% of total serum carotenoids. Vitamin A is formed primarily by the oxygen-dependent central cleavage of beta-carotene and other provitamin A carotenoids. Several carotenoids show enhancement of the immune response, inhibition of mutagenesis, reduction of induced nuclear damage, and protection from various neoplastic events in cells, tissues, and whole animals. Carotenoids also protect against photo-induced tissue damage. Some carotenoids, including beta-carotene, quench highly reactive singlet oxygen under certain conditions and can block free radical-mediated reactions. In epidemiological studies, the intake of carotenoid-rich fruits and vegetables has been correlated with protection from some forms of cancer, particularly lung cancer. Similarly, serum beta-carotene levels have been associated with a decreased chance of developing lung cancer. It must be stressed, however, that these epidemiological associations do not show cause and effect. In this regard, long-term intervention trials with beta-carotene supplements are in progress. Whatever the results of these trials, carotenoids clearly show biological actions in animals distinct from their function as precursors of vitamin A.     

Cassava Intake and Vitamin A Status among Women and Preschool Children in Akwa-Ibom,Nigeria

Background

As part of the HarvestPlus provitamin A-biofortified cassava program in Nigeria we conducted a survey to determine the cassava intake and prevalence of vitamin A deficiency among children 6-59 months and women of childbearing age in the state of Akwa Ibom.

Methods

A cluster-randomized cross-sectional survey was conducted in 2011 in Akwa Ibom, Nigeria. The usual food and nutrient intakes were estimated using a multi-pass 24-hour recall with repeated recall on a subsample. Blood samples of children and women were collected to analyze for serum retinol, serum ferritin, and acute phase proteins as indicators of infection. Vitamin A deficiency was defined as serum retinol <0.70 μmol/L adjusted for infection.

Results

A total of 587 households of a mother-child dyad participated in the dietary intake assessment. Cassava was very widely consumed in Akwa Ibom, mainly as gari or foofoo. Daily cassava consumption frequency was 92% and 95% among children and women, respectively. Mean (±SD) cassava intake (expressed as raw fresh weight) was 348 ± 317 grams/day among children and 940 ± 777 grams/day among women. Intakes of most micronutrients appeared to be adequate with the exception of calcium. Median vitamin A intake was very high both for children (1038 μg RAE/day) and women (2441 μg RAE/day). Red palm oil and dark green leafy vegetables were the main sources of vitamin A in the diet, with red palm oil alone contributing almost 60% of vitamin A intake in women and children. Prevalence of vitamin A deficiency ranged from moderate (16.9 %) among children to virtually non-existent (3.4 %) among women.

Conclusion

Consumption of cassava and vitamin A intake was high among women and children in Akwa Ibom with a prevalence of vitamin A deficiency ranging from moderate in children to non-existent among women. The provitamin A biofortified cassava and other vitamin A interventions should focus dissemination in states where red palm oil is not widely consumed.     

Carotenoid oxygenases: cleave it or leave it

Carotenoid cleavage products (apocarotenoids) are widespread in living organisms and exert key biological functions. In animals, retinoids function as vitamins, visual pigments and signalling molecules. In plants, apocarotenoids play roles as hormones, pigments, flavours, aromas and defence compounds. The first step in their biosynthesis is the oxidative cleavage of a carotenoid catalysed by a non-heme iron oxygenase. A novel family of enzymes, which can cleave different carotenoids at different positions, has been characterized.     

RNA interference-mediated repression of MtCCD1 in mycorrhizal roots of Medicago truncatula causes accumulation of C27 apocarotenoids, shedding light on the functional role of CCD1

Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C₄₀ carotenoid substrates at 9,10 and 9′,10′ positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C₁₃ cyclohexenone and C₁₄ mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C₂₇ apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C₂₇ apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C₂₇ apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C₂₇ → C₁₄ + C₁₃), while the first step (C₄₀ → C₂₇ + C₁₃) may be catalyzed by CCD7 and/or CCD4 inside plastids.     

The Human Enzyme That Converts Dietary Provitamin A Carotenoids to Vitamin A Is a Dioxygenase

β-Carotene 15–15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O₂ and H₂O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either ¹⁶O₂-H₂¹⁸O or ¹⁸O₂-H₂¹⁶O medium for 15 min at 37 °C, and the relative amounts of ¹⁸O-retinal and ¹⁶O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O₂ gas used. Together with the data from ¹⁸O-retinal-H₂¹⁶O and ¹⁶O-retinal-H₂¹⁸O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O₂ into retinal. Thus, BCO1 is a dioxygenase.     

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔI_ami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.     

Monoclonal Antibodies against Aβ42 Fibrils Distinguish Multiple Aggregation State Polymorphisms in Vitro and in Alzheimer Disease Brain

Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process.     

A Role for DNA Polymerase θ in Promoting Replication through Oxidative DNA Lesion,Thymine Glycol,in Human Cells

The biological functions of human DNA polymerase (pol) θ, an A family polymerase, have remained poorly defined. Here we identify a role of polθ in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (TG) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, polymerases κ and ζ function together and mediate error-free TLS, whereas in the other, polθ functions in an error-prone manner. Human polθ is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite TG in human cells. In contrast to TLS mediated by polκ and polζ, in which polζ would elongate the chain from the TG:A base pair formed by polκ action, the ability of polθ alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5′ template base, suggests that the polθ active site can accommodate highly distorting DNA lesions.